Flexible TALEs for an expanded use in gene activation, virulence and scaffold engineering.

Transcription activator-like effectors (TALEs) are bacterial proteins with a programmable DNA-binding domain, which turned them into exceptional tools for biotechnology. TALEs contain a central array of consecutive 34 amino acid long repeats to bind DNA in a simple one-repeat-to-one-nucleotide manner. However, a few naturally occurring aberrant repeat variants break this strict binding mechanism, allowing for the recognition of an additional sequence with a -1 nucleotide frameshift. The limits and implications of this extended TALE binding mode are largely unexplored. Here, we analyse the complete diversity of natural and artificially engineered aberrant repeats for their impact on the DNA binding of TALEs. Surprisingly, TALEs with several aberrant repeats can loop out multiple repeats simultaneously without losing DNA-binding capacity. We also characterized members of the only natural TALE class harbouring two aberrant repeats and confirmed that their target is the major virulence factor OsSWEET13 from rice. In an aberrant TALE repeat, the position and nature of the amino acid sequence strongly influence its function. We explored the tolerance of TALE repeats towards alterations further and demonstrate that inserts as large as GFP can be tolerated without disrupting DNA binding. This illustrates the extraordinary DNA-binding capacity of TALEs and opens new uses in biotechnology.

Altering transcription factor binding reveals comprehensive transcriptional kinetics of a basic gene.

Transcription is a vital process activated by transcription factor (TF) binding. The active gene releases a burst of transcripts before turning inactive again. While the basic course of transcription is well understood, it is unclear how binding of a TF affects the frequency, duration and size of a transcriptional burst. We systematically varied the residence time and concentration of a synthetic TF and characterized the transcription of a synthetic reporter gene by combining single molecule imaging, single molecule RNA-FISH, live transcript visualisation and analysis with a novel algorithm, Burst Inference from mRNA Distributions (BIRD). For this well-defined system, we found that TF binding solely affected burst frequency and variations in TF residence time had a stronger influence than variations in concentration. This enabled us to device a model of gene transcription, in which TF binding triggers multiple successive steps before the gene transits to the active state and actual mRNA synthesis is decoupled from TF presence. We quantified all transition times of the TF and the gene, including the TF search time and the delay between TF binding and the onset of transcription. Our quantitative measurements and analysis revealed detailed kinetic insight, which may serve as basis for a bottom-up understanding of gene regulation.

Engineered TALE Repeats for Enhanced Imaging-Based Analysis of Cellular 5-Methylcytosine.

Transcription-activator-like effectors (TALEs) are repeat-based, programmable DNA-binding proteins that can be engineered to recognize sequences of canonical and epigenetically modified nucleobases. Fluorescent TALEs can be used for the imaging-based analysis of cellular 5-methylcytosine (5 mC) in repetitive DNA sequences. This is based on recording fluorescence ratios from cell co-stains with two TALEs: an analytical TALE targeting the cytosine (C) position of interest through a C-selective repeat that is blocked by 5 mC, and a control TALE targeting the position with a universal repeat that binds both C and 5 mC. To enhance this approach, we report herein the development of novel 5 mC-selective repeats and their integration into TALEs that can replace universal TALEs in imaging-based 5 mC analysis, resulting in a methylation-dependent response of both TALEs. We screened a library of size-reduced repeats and identified several 5 mC binders. Compared to the 5 mC-binding repeat of natural TALEs and to the universal repeat, two repeats containing aromatic residues showed enhancement of 5 mC binding and selectivity in cellular transcription activation and electromobility shift assays, respectively. In co-stains of cellular SATIII DNA with a corresponding C-selective TALE, this selectivity results in a positive methylation response of the new TALE, offering perspectives for studying 5 mC functions in chromatin regulation by in situ imaging with increased dynamic range.

Chimerization Enables Gene Synthesis and Lentiviral Delivery of Customizable TALE-Based Effectors.

Designer effectors based on the DNA binding domain (DBD) of Xanthomonas transcription activator-like effectors (TALEs) are powerful sequence-specific tools with an excellent reputation for their specificity in editing the genome, transcriptome, and more recently the epigenome in multiple cellular systems. However, the repetitive structure of the TALE arrays composing the DBD impedes their generation as gene synthesis product and prevents the delivery of TALE-based genes using lentiviral vectors (LVs), a widely used system for human gene therapy. To overcome these limitations, we aimed at chimerizing the DNA sequence encoding for the TALE-DBDs by introducing sufficient diversity to facilitate both their gene synthesis and enable their lentiviral delivery. To this end, we replaced three out of 17 Xanthomonas TALE repeats with TALE-like units from the bacterium Burkholderia rhizoxinica. This was combined with extensive codon variation and specific amino acid substitutions throughout the DBD in order to maximize intra- and inter-repeat sequence variability. We demonstrate that chimerized TALEs can be easily generated using conventional Golden Gate cloning strategy or gene synthesis. Moreover, chimerization enabled the delivery of TALE-based designer nucleases, transcriptome and epigenome editors using lentiviral vectors. When delivered as plasmid DNA, chimerized TALEs targeting the CCR5 and CXCR4 loci showed comparable activities in human cells. However, lentiviral delivery of TALE-based transcriptional activators was only successful in the chimerized form. Similarly, delivery of a chimerized CXCR4-specific epigenome editor resulted in rapid silencing of endogenous CXCR4 expression. In conclusion, extensive codon variation and chimerization of TALE-based DBDs enables both the simplified generation and the lentiviral delivery of designer TALEs, and therefore facilitates the clinical application of these tools to precisely edit the genome, transcriptome and epigenome.

Structural Insights into the Specific Recognition of 5-methylcytosine and 5-hydroxymethylcytosine by TAL Effectors.

Transcription activator-like effectors (TALEs) recognize DNA through repeat-variable diresidues (RVDs), and TALE-DNA interactions are sensitive to DNA modifications. Our previous study deciphered the recognition of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) by TALEs. Here, we report seven crystal structures of TALE-DNA complexes. The 5mC-specific RVD HA recognizes 5mC through van der Waals interactions and exhibits highly similar loop conformation to natural RVDs. The degenerate RVD RG contacts 5mC and 5hmC via van der Waals interactions as well; however, its loop conformation differs significantly. The loop conformations of universal RVD R* and 5hmC-specific RVD Q* are similar to that of RG, while the interactions of R* with C/5mC/5hmC and Q* with 5hmC are mediated by waters. Together, our findings illustrate the molecular basis for the specific recognition of 5mC and 5hmC by multiple noncanonical TALEs and provide insights into the plasticity of the TALE RVD loops.

Context and number of noncanonical repeat variable diresidues impede the design of TALE proteins with improved DNA targeting.

Transcription activator-like effector (TALE) proteins have been used extensively for targeted binding of fusion proteins to loci of interest in (epi)genome engineering. Such approaches typically utilize four canonical TALE repeat variable diresidue (RVD) types, corresponding to the identities of two key amino acids, to target each nucleotide. Alternate RVDs with improved specificity are desired. Here, we focused on seven noncanonical RVDs that have been suggested to have improved specificity for their target nucleotides. We used custom protein binding microarrays to characterize the DNA-binding activity of 65 TALEs containing these alternate or corresponding canonical RVDs at multiple positions to ~5,000 unique DNA sequences per protein. We found that none of the noncanonical thymine-targeting RVDs displayed stronger preference for thymine than did the canonical RVD. Of the noncanonical RVDs with putatively improved specificity for guanine, only EN and NH showed greater discrimination of guanine over adenine. This improved specificity, however, comes at a cost: more substitutions of a noncanonical RVD for a canonical RVD generally decreased the protein's DNA-binding activity. Our results highlight the need to investigate RVD-nucleotide specificities in multiple protein contexts and suggest that a balance between canonical and noncanonical RVDs is needed to build TALEs with improved specificity.

Truncated TALE-FP as DNA Staining Dye in a High-salt Buffer.

Large DNA molecules are a promising platform for in vitro single-molecule biochemical analysis to investigate DNA-protein interactions by fluorescence microscopy. For many studies, intercalating fluorescent dyes have been primary DNA staining reagents, but they often cause photo-induced DNA breakage as well as structural deformation. As a solution, we previously developed several fluorescent-protein DNA-binding peptides or proteins (FP-DBP) for reversibly staining DNA molecules without structural deformation or photo-induced damage. However, they cannot stain DNA in a condition similar to a physiological salt concentration that most biochemical reactions require. Given these concerns, here we developed a salt-tolerant FP-DBP: truncated transcription activator-like effector (tTALE-FP), which can stain DNA up to 100 mM NaCl. Moreover, we found an interesting phenomenon that the tTALE-FP stained DNA evenly in 1 × TE buffer but showed AT-rich specific patterns from 40 mM to 100 mM NaCl. Using an assay based on fluorescence resonance energy transfer, we demonstrated that this binding pattern is caused by a higher DNA binding affinity of tTALE-FP for AT-rich compared to GC-rich regions. Finally, we used tTALE-FP in a single molecule fluorescence assay to monitor real-time restriction enzyme digestion of single DNA molecules. Altogether, our results demonstrate that this protein can provide a useful alternative as a DNA stain over intercalators.

Programmable Protein-DNA Cross-Linking for the Direct Capture and Quantification of 5-Formylcytosine.

5-Formylcytosine (5fC) is an epigenetic nucleobase of mammalian genomes that occurs as intermediate of active DNA demethylation. 5fC uniquely interacts and reacts with key nuclear proteins, indicating functions in genome regulation. Transcription-activator-like effectors (TALEs) are repeat-based DNA binding proteins that can serve as probes for the direct, programmable recognition and analysis of epigenetic nucleobases. However, no TALE repeats for the selective recognition of 5fC are available, and the typically low genomic levels of 5fC represent a particular sensitivity challenge. We here advance TALE-based nucleobase targeting from recognition to covalent cross-linking. We report TALE repeats bearing the ketone-amino acid p-acetylphenylalanine (pAcF) that universally bind all mammalian cytosine nucleobases, but selectively form diaminooxy-linker-mediated dioxime cross-links to 5fC. We identify repeat-linker combinations enabling single CpG resolution, and demonstrate the direct quantification of 5fC levels in a human genome background by covalent enrichment. This strategy provides a new avenue to expand the application scope of programmable probes with selectivity beyond A, G, T and C for epigenetic studies.

Assembly of TALE-based DNA scaffold for the enhancement of exogenous multi-enzymatic pathway.

Synthetic scaffold systems, which exhibit enzyme clustering effect, have been considered as an important parallel approach for metabolic flux control and pathway enhancement. Here, we described an improved DNA-based scaffold system for synthetic tri-enzymatic pathway in Escherichia coli. With plasmid DNA serving as scaffold and exogenous enzymes fused with rationally designed transcription activator-like effectors (TALEs), our approach successfully clustered three TALE-fused enzymes and significantly increased the production of a mevalonate-producing tri-enzymatic pathway with the optimized scaffold structure and plasmid copy number. These results further suggested the scalability and robustness of the TALE-based scaffold system, and we can assume that it can be used on numerous multi-enzyme metabolic pathways due to its programmable features.

Design, Construction, and Application of Transcription Activation-Like Effectors.

Transcription activator-like effectors (TALEs) are modular proteins derived from the plant Xanthomonas sp. pathogen that can be designed to target unique DNA sequences following a simple cipher. Customized TALE proteins can be used in a variety of molecular applications that include gene editing and transcriptional modulation. Presently, we provide a brief primer on the design and construction of TALEs. TALE proteins can be fused to a variety of different effector domains that alter the function of the TALE upon binding. This flexibility of TALE design and downstream effect may offer therapeutic applications that are discussed in this section. Finally, we provide a future perspective on TALE technology and what challenges remain for successful translation of gene-editing strategies to the clinic.

Fast and global detection of periodic sequence repeats in large genomic resources.

Periodically repeating DNA and protein elements are involved in various important biological events including genomic evolution, gene regulation, protein complex formation, and immunity. Notably, the currently used genome editing tools such as ZFNs, TALENs, and CRISPRs are also all associated with periodically repeating biomolecules of natural organisms. Despite the biological importance of periodically repeating sequences and the expectation that new genome editing modules could be discovered from such periodical repeats, no software that globally detects such structured elements in large genomic resources in a high-throughput and unsupervised manner has been developed. We developed new software, SPADE (Search for Patterned DNA Elements), that exhaustively explores periodic DNA and protein repeats from large-scale genomic datasets based on k-mer periodicity evaluation. With a simple constraint, sequence periodicity, SPADE captured reported genome-editing-associated sequences and other protein families involving repeating domains such as tetratricopeptide, ankyrin and WD40 repeats with better performance than the other software designed for limited sets of repetitive biomolecular sequences, suggesting the high potential of this software to contribute to the discovery of new biological events and new genome editing modules.

Visualization of Genomic Loci in Living Cells with BiFC-TALE.

Tracking the dynamics of genomic loci is essential for understanding a variety of cellular processes. However, earlier methods have all suffered from a low signal-to-background ratio (SBR), mainly caused by the background fluorescence from diffuse full-length fluorescent proteins in the nucleus. We have developed a novel method (BiFC-TALE) for labeling and tracking genomic loci in live mammalian cells, combining bimolecular fluorescence complementation (BiFC) and transcription activator-like effector (TALE) technologies. Since only the sequences-targeted BiFC fragments can be pulled together by TALE modules to recombine intact fluorescent proteins, the background fluorescence in the living nucleus can be largely reduced, which significantly improves SBR. Using telomere and centromere labeling as examples, this unit describes in detail the design and implementation of BiFC-TALE system. © 2018 by John Wiley & Sons, Inc.

Sequence-specific 5mC detection in live cells based on the TALE-split luciferase complementation system.

We established a method for converting TALE-DNA binding to luminescence, by combining a TALE and a split luciferase system. Furthermore, using a methylation-sensitive TALE, sequence-specific 5mC detection of genomic DNA was achieved in live cells. This study provides a new strategy for exploring the biological functions of 5mC.

Engineering altered protein-DNA recognition specificity.

Protein engineering is used to generate novel protein folds and assemblages, to impart new properties and functions onto existing proteins, and to enhance our understanding of principles that govern protein structure. While such approaches can be employed to reprogram protein-protein interactions, modifying protein-DNA interactions is more difficult. This may be related to the structural features of protein-DNA interfaces, which display more charged groups, directional hydrogen bonds, ordered solvent molecules and counterions than comparable protein interfaces. Nevertheless, progress has been made in the redesign of protein-DNA specificity, much of it driven by the development of engineered enzymes for genome modification. Here, we summarize the creation of novel DNA specificities for zinc finger proteins, meganucleases, TAL effectors, recombinases and restriction endonucleases. The ease of re-engineering each system is related both to the modularity of the protein and the extent to which the proteins have evolved to be capable of readily modifying their recognition specificities in response to natural selection. The development of engineered DNA binding proteins that display an ideal combination of activity, specificity, deliverability, and outcomes is not a fully solved problem, however each of the current platforms offers unique advantages, offset by behaviors and properties requiring further study and development.

Complete, Programmable Decoding of Oxidized 5-Methylcytosine Nucleobases in DNA by Chemoselective Blockage of Universal Transcription-Activator-Like Effector Repeats.

5-Methylcytosine (5mC) and its oxidized derivatives are regulatory elements of mammalian genomes involved in development and disease. These nucleobases do not selectively modulate Watson-Crick pairing, preventing their programmable targeting and analysis by traditional hybridization probes. Transcription-activator-like effectors (TALEs) can be engineered for use as programmable probes with epigenetic nucleobase selectivity. However, only partial selectivities for oxidized 5mC have been achieved so far, preventing unambiguous target binding. We overcome this limitation by destroying and re-inducing nucleobase selectivity in TALEs via protein engineering and chemoselective nucleobase blocking. We engineer cavities in TALE repeats and identify a cavity that accommodates all eight human DNA nucleobases. We then introduce substituents with varying size, flexibility, and branching degree at each oxidized 5mC. Depending on the nucleobase, substituents with distinct properties effectively block TALE-binding and induce full nucleobase selectivity in the universal repeat. Successful transfer to affinity enrichment in a human genome background indicates that this approach enables the fully selective detection of each oxidized 5mC in complex DNA by programmable probes.

Selective recognition of N4-methylcytosine in DNA by engineered transcription-activator-like effectors.

The epigenetic DNA nucleobases 5-methylcytosine (5mC) and N4-methylcytosine (4mC) coexist in bacterial genomes and have important functions in host defence and transcription regulation. To better understand the individual biological roles of both methylated nucleobases, analytical strategies for distinguishing unmodified cytosine (C) from 4mC and 5mC are required. Transcription-activator-like effectors (TALEs) are programmable DNA-binding repeat proteins, which can be re-engineered for the direct detection of epigenetic nucleobases in user-defined DNA sequences. We here report the natural, cytosine-binding TALE repeat to not strongly differentiate between 5mC and 4mC. To engineer repeats with selectivity in the context of C, 5mC and 4mC, we developed a homogeneous fluorescence assay and screened a library of size-reduced TALE repeats for binding to all three nucleobases. This provided insights into the requirements of size-reduced TALE repeats for 4mC binding and revealed a single mutant repeat as a selective binder of 4mC. Employment of a TALE with this repeat in affinity enrichment enabled the isolation of a user-defined DNA sequence containing a single 4mC but not C or 5mC from the background of a bacterial genome. Comparative enrichments with TALEs bearing this or the natural C-binding repeat provides an approach for the complete, programmable decoding of all cytosine nucleobases found in bacterial genomes.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.

Designer epigenome modifiers enable robust and sustained gene silencing in clinically relevant human cells.

Targeted modulation of gene expression represents a valuable approach to understand the mechanisms governing gene regulation. In a therapeutic context, it can be exploited to selectively modify the aberrant expression of a disease-causing gene or to provide the target cells with a new function. Here, we have established a novel platform for achieving precision epigenome editing using designer epigenome modifiers (DEMs). DEMs combine in a single molecule a DNA binding domain based on highly specific transcription activator-like effectors (TALEs) and several effector domains capable of inducing DNA methylation and locally altering the chromatin structure to silence target gene expression. We designed DEMs to target two human genes, CCR5 and CXCR4, with the aim of epigenetically silencing their expression in primary human T lymphocytes. We observed robust and sustained target gene silencing associated with reduced chromatin accessibility, increased promoter methylation at the target sites and undetectable changes in global gene expression. Our results demonstrate that DEMs can be successfully used to silence target gene expression in primary human cells with remarkably high specificity, paving the way for the establishment of a potential new class of therapeutics.

Zinc Fingers, TALEs, and CRISPR Systems: A Comparison of Tools for Epigenome Editing.

The completion of genome, epigenome, and transcriptome mapping in multiple cell types has created a demand for precision biomolecular tools that allow researchers to functionally manipulate DNA, reconfigure chromatin structure, and ultimately reshape gene expression patterns. Epigenetic editing tools provide the ability to interrogate the relationship between epigenetic modifications and gene expression. Importantly, this information can be exploited to reprogram cell fate for both basic research and therapeutic applications. Three different molecular platforms for epigenetic editing have been developed: zinc finger proteins (ZFs), transcription activator-like effectors (TALEs), and the system of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins. These platforms serve as custom DNA-binding domains (DBDs), which are fused to epigenetic modifying domains to manipulate epigenetic marks at specific sites in the genome. The addition and/or removal of epigenetic modifications reconfigures local chromatin structure, with the potential to provoke long-lasting changes in gene transcription. Here we summarize the molecular structure and mechanism of action of ZF, TALE, and CRISPR platforms and describe their applications for the locus-specific manipulation of the epigenome. The advantages and disadvantages of each platform will be discussed with regard to genomic specificity, potency in regulating gene expression, and reprogramming cell phenotypes, as well as ease of design, construction, and delivery. Finally, we outline potential applications for these tools in molecular biology and biomedicine and identify possible barriers to their future clinical implementation.

Designing Epigenome Editors: Considerations of Biochemical and Locus Specificities.

The advent of locus-specific protein recruitment technologies has enabled a new class of studies in chromatin biology. Epigenome editors enable biochemical modifications of chromatin at almost any specific endogenous locus. Their locus specificity unlocks unique information including the functional roles of distinct modifications at specific genomic loci. Given the growing interest in using these tools for biological and translational studies, there are many specific design considerations depending on the scientific question or clinical need. Here we present and discuss important design considerations and challenges regarding the biochemical and locus specificities of epigenome editors. These include how to account for the complex biochemical diversity of chromatin; control for potential interdependency of epigenome editors and their resultant modifications; avoid sequestration effects; quantify the locus specificity of epigenome editors; and improve locus specificity by considering concentration, affinity, avidity, and sequestration effects.

Generation of TALE-Based Designer Epigenome Modifiers.

Manipulation of gene expression can be facilitated by editing the genome or the epigenome. Precise genome editing is traditionally achieved by using designer nucleases which are generally exploited to eliminate a specific gene product. Upon the introduction of a site-specific DNA double-strand break (DSB) by the nuclease, endogenous DSB repair mechanisms are in turn harnessed to induce DNA sequence changes that can result in target gene inactivation. Minimal off-target effects can be obtained by endowing designer nucleases with the highly specific DNA-binding domain (DBD) derived from transcription activator-like effectors (TALEs). In contrast, epigenome editing allows gene expression control without inducing changes in the DNA sequence by specifically altering epigenetic marks, as histone tails modifications or DNA methylation patterns within promoter or enhancer regions. Importantly, this approach allows both up- and downregulation of the target gene expression, and the effect is generally reversible. TALE-based designer epigenome modifiers combine the high specificity of TALE-derived DBDs with the power of epigenetic modifier domains to induce fast and long-lasting changes in the epigenetic landscape of a target gene and control its expression. Here we provide a detailed description for the generation of TALE-based designer epigenome modifiers and of a suitable reporter cell line to easily monitor their activity.